This project has two primary aims. The first aim is to understand how proteins which are secreted or inserted into membranes are targeted to transport sites in the endoplasmic reticulum (ER) or, equivalently, the bacterial inner membrane (IM). In particular we are investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its ER-bound receptor in this process. Previous studies have shown that SRP recognizes nascent polypeptide chains containing "signal sequences" and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor (SR). We plan to elucidate the mechanism by which SRP recognizes the highly diverse family of signal sequences found on different proteins and then releases them at the surface of the ER in a regulated fashion. By studying this problem we expect to obtain insight into the function and regulation of proteins that possess broad substrate specificity. The second aim of the project is to elucidate the function of a universal protein conducting channel or "translocon" that is found in both the ER and the IM. We are particularly interested in understanding how the translocon catalyzes two related but seemingly distinct processes, namely the complete transfer of presecretory proteins across a membrane and the integration of membrane proteins into a lipid bilayer. In addition, we would like to determine why the core of the translocon is evolutionarily conserved, but the peripheral subunits are not. We expect that these experiments will provide significant insight into the function of membrane-bound channels. With the advent of the genomic era it has become clear that SRP and the SR are extraordinarily well conserved throughout evolution. A few years ago we demonstrated that although molecular chaperones target most periplasmic and outer membrane proteins to the bacterial IM, SRP targets integral membrane proteins to the secretory pathway. The discovery of a functional link between eukaryotic and prokaryotic SRP has now made it possible to use E. coli as a simple, genetically tractable model organism to study the SRP pathway. Recently we have turned to the E. coli system to explain some unusual properties of two homologous GTPases in the SRP pathway, the SRP signal peptide binding subunit (SRP54) and the SR a-subunit (SRa). Previous work has shown that an interaction between these two GTPases plays an essential role in the targeting reaction. Unlike other GTPases that have been studied to date, these two proteins reciprocally regulate each other's GTPase activity. The GTPase domain of both proteins abuts a unique "N domain" that appears to facilitate the binding of external ligands such as signal peptides. To examine the relationship between the unusual regulation and unique architecture of the SRP pathway GTPases, we mutated an invariant glycine in E. coli SRP54 and SRa orthologs ("Ffh" and "FtsY", respectively) that resides at the N-GTPase domain interface. We found that a G257A mutation in Ffh produced a lethal phenotype. The mutation did not significantly affect protein stability, signal peptide binding or GTPase activity, but severely reduced interaction with FtsY. Likewise, mutation of FtsY glycine 455 produced growth defects and inhibited interaction with Ffh. The data suggest that Ffh and FtsY interact only in a "primed" conformation which requires interdomain communication. Based on these results, we have proposed that the distinctive features of the SRP pathway GTPases evolved to ensure that SRP and the SR engage external ligands before interacting with each other.